RENAL DATA FROM ASIA–AFRICA
|Year : 2018 | Volume
| Issue : 4 | Page : 930-938
|Spectrum of renal allograft biopsy: A five-year experience at a tertiary care center of Eastern India
Malagouda R Patil1, Smita Subhash Divyaveer2, Chetan Mahajan2, Arpita Roy Choudhury2, Sanjay Dasgupta2, Dipankar Sarkar2, Harmeet Riyait2, Anila Abraham3, Rajendra Pandey2
1 Department of Nephrology, Karnataka Institute of Medical Sciences, Hubli, India
2 Department of Nephrology, Institute of Post-Graduate Medical Education and Research and Seth Sukhlal Karnani Memorial Hospital, Kolkata, India
3 Center for Renal and Urological Pathology, Chennai, Tamil Nadu, India, India
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|Date of Submission||24-Jul-2017|
|Date of Decision||09-Oct-2017|
|Date of Acceptance||23-Oct-2017|
|Date of Web Publication||28-Aug-2018|
| Abstract|| |
Renal allograft dysfunction (RAD) can have myriad causes and presentations. Allograft biopsy remains the gold standard for optimum management. This is a retrospective study carried out at a tertiary care institute from August 2011 to March 2016. Details of the renal allograft biopsy requisitions were recorded and analyzed. Two hundred and two patients had undergone kidney transplantation (KT) during the study period. One hundred and twenty-six had undergone renal biopsy for RAD. The acute asymptomatic rise of serum creatinine was the most common clinical presentation (47.61%) followed by chronic RAD (CRAD) (19.84%), proteinuria (15.87%), immediate graft dysfunction (10.31%), and persistent active urinary sediments (6.34%) in that order. The incidence of delayed graft function was 1.98%. The overall incidence of biopsy-proven rejection was 8.41% within oneyear and 8.91% beyond oneyear of transplant. Acute cellular rejection (ACR) [with or without antibody-mediated rejection (AMR)] was found in 65%; AMR was found in 40% and 15% had both ACR and AMR. Borderline acute cell-mediated rejection was found in 22.5% of biopsies. CRAD was due to chronic rejection and chronic calcineurin inhibitor toxicity in only about one-fourth of the cases. Incidence of glomerulo-nephritis was 10.89% and most of these occurred two years after KT. Renal allograft biopsy was associated with minor complications in 3.17% of cases. Clinical presentations do not reliably distinguish the various causes of RAD. Allograft biopsy is a mainstay in the diagnosis of RAD and is safe. Results of live donor first transplantation using complement-dependent cytotoxi-city crossmatch are comparable to those programs using newer methods like solid-phase assays. However, the direct comparison of these results with other studies may not be completely applicable.
|How to cite this article:|
Patil MR, Divyaveer SS, Mahajan C, Choudhury AR, Dasgupta S, Sarkar D, Riyait H, Abraham A, Pandey R. Spectrum of renal allograft biopsy: A five-year experience at a tertiary care center of Eastern India. Saudi J Kidney Dis Transpl 2018;29:930-8
|How to cite this URL:|
Patil MR, Divyaveer SS, Mahajan C, Choudhury AR, Dasgupta S, Sarkar D, Riyait H, Abraham A, Pandey R. Spectrum of renal allograft biopsy: A five-year experience at a tertiary care center of Eastern India. Saudi J Kidney Dis Transpl [serial online] 2018 [cited 2019 Dec 6];29:930-8. Available from: http://www.sjkdt.org/text.asp?2018/29/4/930/239638
| Introduction|| |
Renal biopsy remains the “gold standard” for the diagnosis of renal allograft dysfunction (RAD). According to available studies, the results of a renal allograft biopsy changed the clinical diagnosis in 27%–46% of patients and therapy in 38%–83%, even after the 1st year.,, The arrival of c4d staining of the renal allo-graft biopsy and its wide availability has been a major development in the diagnosis and hence the management of RAD. Although encouraging data for the diagnosis of rejection in kidney transplantation (KT) by noninvasive means is emerging, these are yet to be established in standard clinical settings. The data on the spectrum of renal allograft biopsies and its clinical correlation in the modern era of immunosuppression and recent updated classification of graft rejection are relatively few. The newer crossmatching techniques are not widely available and/or economically accessible in many parts of India. Besides, the outcome of renal transplantation varies between different centers hence the need to analyze the biopsy registry in our settings is of paramount importance. Another important aspect of this study is to analyze the clinico-pathological scenario in the light of the updated Banff classification.
| Materials and Methods|| |
This is a retrospective observational singlecenter study of the biopsies performed for diagnosis of RAD between August 2011 and March 2016 (n=126) at the Institute of Postgraduate Medical Education and Research (IPGMER), Kolkata. For collection of data the registry of the institute and renal allograft biopsy (Bx) requisitions containing an indication of biopsy, clinical and laboratory parameters at the time of biopsy, duration since transplant were reviewed. Descriptive statistics have been used for analysis such as mean and standard deviation (SD).
Renal biopsy procedure
As a protocol, two cores of renal graft tissue were obtained with automated biopsy gun (16G ×10cm) under real-time ultrasonography guidanceand subjected to light microscopy (using hematoxylinand eosin, periodic acid-Schiff stain, methenamine silver stain, and trichrome stain) and immunofluorescence (IF). IF included staining for c4d, IgG, IgM, IgA, C3, C1q, kappa, and lambda light chains. All biopsy samples were analyzed by a single nephropathologist. When thought necessary by the treating physician electron microscopy was done using the paraffin-embedded tissue. All patients were admitted for a day unless any complications occurred that needed further hospital stay. The complications were noted retrospectively from available records.
Selection of donors and immunosuppression protocol
IPGMER, Kolkata is the largest tertiary care government center in Eastern India. Most of the renal transplantations are from live donors. The necessary clearance for kidney donation was obtained in all cases from the institutional/ state level transplant committee as per Government norms. Only ABO-compatible donors were selected. Immunosuppression is started two days before transplantation with tacrolimus and mycophenolate (MPA). Pulse methylprednisolone is given for three days including the day of transplant. Induction regime with basiliximab was given in only unrelated KT from August 2011 to March 2012. All patients who underwent transplantation between April 2012 and March 2016 had received induction with either basiliximab or rabbit anti-thymocyte globulin (rATG). MPA was switched to azathioprine in some patients who developed intolerable side effects to MPA. Oral prednisolone was progressively reduced to 7.5 mg/day at the end of 3rd month post-KT, and the tacrolimus dose was adjusted to maintain blood levels around 9–12 ng/dL in the first three months; 5–7 ng/dL during months 3–6 and 3–5 ng/dL subsequently. However, the dosage of each of these maintenance drugs was adjusted and tailored as indicated clinically. As per the institute's protocol patients were advised to follow-up once a week for 1st month after discharge; once in two weeks in 2nd to 3rd months; once monthly from month three to six and three monthly thereon. The follow-up protocol was individualized and adjusted as clinically indicated.
Indications of percutaneous renal allograft biopsy
- Increase in the serum creatinine (SCr) level to >25% above baseline over days to weeks without any associated signs or symptoms, for example, including fever, gross hematuria, renal pain, and oliguria (i.e., asymptomatic rise of SCr)
- Immediate graft function (delayed or slow graft dysfunction)
- Chronic renal graft dysfunction (CRAD), i.e., rise of creatinine over a period of months
- Abnormal urinalysis with either glome-rular hematuria and/or proteinuria.
No protocol biopsies were done.
| Results|| |
The total number of renal transplants done was 202 (44 ± 6 per year) between August 2011 and March 2016. The native kidney disease (NKD) was unknown in most of the patients, but urine examination was suggestive of chronic glomerulonephritis (GN). Diabetic nephropathy (DN) was NKD in 12.37% (n=25) of cases. Obstructive uropathy due to pelviureteric junction obstruction or posterior urethral valve was the NKD in 4.95% (n=10) Out of the 202 transplants that were done in the study period, 22 live-related renal allograft recipients (RAR) received no induction (between August 2011 and March 2012, a period when induction was given only in unrelated cases), whereas all others received induction in the form of either basiliximab (20 mg, 1 dose each on day of transplant and 20 mg and on the postoperative day 4) or rATG (0.7 mg/kg/day for 7 days). rATG was given as induction agent for 17 patients (rATG associated with higher incidence of sepsis and similar rates of rejection: unpublished data from our center). One hundred and sixty-three patients received basiliximab as induction agent irrespective of whether donor was related or not. Data of 126 graft biopsies wereincluded for analysis. All except one (0.79%) had adequate renal tissue. A total of 106 (84.12%) renal allograft recipients (RAR) were male and 20 (15.87%) were female. Donors were related in 55.55% (n=70) of cases and unrelated in 44.44% (n=56). Majority of unrelated donors were spouse of recipients 34.92% (n=44). The ratio of related and unrelated donors was 1.25:1. Average age of RAR at the time of biopsy was 32.93 (SD ±8.87) years. Range of age at the time of biopsy was 13–57 years. Only three patients were <18 years andthree others were more than 50 years of age at the time of biopsy. Sixty (47.61%) biopsies were done in the 1st year of posttransplant period and 66 (52.38%) were done beyond one year of post-transplant period. The indications of biopsy in each subgroup are shown in [Table 1]. Both delayed and slow graft dysfunction were included in one group as immediate graft dysfunction (IGD). The overall incidence of delayed graft function (DGF), i.e., requirement of dialysis within one week of KT was 1.98% (n= 4).
|Table 1: Indications of biopsy according to the timing of the biopsy (less than or more than 1year) and overall.|
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The distribution of the biopsy findings are shown in [Table 2]. Out of the total 202 RAR who underwent KT, rejection was found as a cause graft dysfunction in 8.41% and 8.91% within the 1st year and beyond 1st year of transplantation, respectively. Of all the biopsies done for graft dysfunction (n=126), rejection was found in a total of 27.77%; of these 13.49% (n=17) occurred within one year of transplant and 14.28% (n=18) beyond one year. If acute tubular injury was present along with findings suggestive of calcineurin inhibitor (CNI) toxicity, in the absence of any other cause (as mentioned in biopsy requisition), these were considered to be due to acute CNI toxicity. One case had suspicious borderline acute cell-mediated rejection (CMR) along with IgA nephropathy. This case was included in GN group.
|Table 2: The distribution of biopsy findings according to the timing of biopsy (less than or more than 1 year).|
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The biopsy diagnoses of GN and rejection were more common after the 1st year of transplantation.
In the early posttransplantation period, i.e., within six months, 41 patients had undergone biopsy and acute tubular necrosis (ATN) was the most frequent diagnosis [Figure 1].
|Figure 1: Frequency of biopsy diagnosis in early posttransplant period.|
ATN: Acute tubular necrosis, AMR: Antibodymediated rejection, CMR: Cell-mediated rejection, GN: Glomerulonephritis, CNI: Calcinuerin inhibitor.
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We reviewed biopsy findings about the various clinical presentations to analyze the comparative frequencies of each biopsy diagnosis with a particular clinical presentation. [Figure 2] shows the frequency of various biopsy findings in patients who presented with acute asymptomatic rise of SCr. Among these, “others” included three inconclusive biopsies, cytomegalovirus nephropathy, and pyelonephritis in one each; two had chronic changes (diffuse glomerulosclerosis, interstitial fibrosis, and tubular atrophy) not attributable to any particular cause. [Figure 3] shows the frequency of various biopsy findings in patients who presented with CRAD. Of these, six patients included in “others” were five cases had chronic changes not attributable to any particular cause, one was inconclusive, one had ischemic glo-merular changes with chronicity and one had acute kidney injury (AKI) with C4d negative transplant glomerulopathy (TG). [Figure 4] shows the frequency of various biopsy findings in patients who presented with IGD. [Figure 5] shows the frequency of various biopsy findings in patients who presented with proteinuria. Of these, four patients included in “others,” three had C4d negative TG and one had chronic changes not attributable to any particular cause. [Figure 6] shows the frequency of various biopsy findings in patients who presented with active persistent urinary sediments. In the GN group, focal and segmental glomerulosclerosis (FSGS) was the most frequent diagnosis, occurring in nine biopsies. Four patients showed features of membranous nephropathy, and two patients had membrano-proliferative GN type I, two had IgA nephropathy (IgAN), one patient had crescentic GN, and one had mesangioproliferative GN (all stains negative in immunofluorescence). Features of thrombotic microangiopathy were seen in three biopsies. Among the biopsies done in the early post-KT period, only one patient had GN at three months post-KT (which was FSGS). The average months of the occurrence of individual GN is shown in [Table 3].
|Figure 2: Clinical presentation as the asymptomatic acute rise of serum creatinine.|
ATN: Acute tubular necrosis, CNI: Calcineurin inhibitor, GN: Glomerulonephritis.
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|Figure 3: Clinical presentation as chronic renal allograft dysfunction.|
CNI: Calcinuerin inhibitor, GN: Glomerulonephritis, ATN: Acute tubular necrosis.
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|Figure 4: Clinical presentation as immediate graft function.|
ATN: Acute tubular necrosis, CNI: Calcineurin inhibitor.
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|Figure 5: Clinical presentation as proteinuria.|
GN: Glomerulonephritis, ATN: Acute tubular necrosis.
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|Figure 6: Clinical presentation as persistent active urinary sediments.|
ATN: Acute tubular necrosis, GN: Glomerulonephritis, CNI: Calcineurin inhibitor
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|Table 3: Average duration of occurrence of glomerulonephritis posttransplant in months.|
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There were no clinically apparent complications of renal biopsy in 120 (95.23%) patients. Four (3.17%) patients had gross hematuria which subsided spontaneously within 6–8 h. Two (1.58%) patients had perinephrichematoma. All complications were managed conservatively and none required any surgical/ endovascular intervention. None required blood transfusion for bleeding complications. No patient had graft loss or immediate worsening of graft function after the procedure.
| Discussion|| |
The data on the spectrum of renal allograft biopsy findings from the eastern part of India are very scarce. This study analyzes the incidence of the various causes and presentations of graft dysfunction in our clinical setting. The interpretation of results of any biopsy registry essentially needs a comprehensive consideration of the transplantation protocols, characteristics of the pool of renal allograft recipients and donors and the prevailing socioeconomic conditions. DN is the most common cause of CKD in our population, a finding similar to that of the developed countries. However, CKD of undetermined etiology and chronic GN are the second and third most common causes. Majority of patients presenting to our instituteare from low socioeconomic group, younger and more frequently have CKD of unknown etiology. Despite DN being the most common cause of CKD, patients with DN did not constitute a significant proportion of our RAR probably because they often did not present to our KT clinic due to comorbidities or due to socioeconomic conditions which are less favorable for optimum care of the elderly. The average age of patients at the time of biopsy in our study was 32.93 (SD ±8.87) years. This is similar to the reports from other developing countries.,, In the developed world olderRAR constitute a significant proportion of KT recipients (KTR). Recipient age has been found in most but not all studies to be associated with poor graft outcome both in developed and developing countries.,,, Relatively, more robust immunity, higher prevalence of some glomerulonephritis, and poorer compliance to medication may be the factors involved in influencing the graft dysfunction in young RAR. The number of male RAR was far more than females. The male:female ratio being 5.3:1. This skewed ratio has been also described in other manuscripts from developing countries. Majority of our patients received induction with basiliximab irrespective of whether the donor was related or not. The number of related donor KT was slightly more than unrelated. This is similar to data from other developing countries where emotionally related (biologically unrelated) spousal donors constitute a significant pool in live donor transplant programs.
The incidence of rejection was 8.41% within the 1st year which is close to that reported from the developed countries. About 8.91% had rejection beyond one year of KT. Of all the biopsies done for graft dysfunction, rejection was found in a total 27.77% of cases. Acute cellular rejection (with or without ABMR) was found in 65%; AMR was found in 40% and 15% had both ACR and AMR. Borderline acute CMR was found in 22.5% of biopsies. The overall incidence of ABMR (with c4d positivity) was 47.5%. These findings are closer to that found in other parts of India., The proportion of ABMR in Bx has been variable in different reports and studies., Moreover, ACR and AMR can coexist. Recognition of c4d negative ABMR is another significant development in the classification of rejection. Due to these changes in classification, direct comparisons between studies and registries from earlier decades would be inaccurate. Furthermore, comparisons with older studies may not be appropriate as the transplant protocols used earlier were different from the ones that are currently followed widely. While therapeutic drug monitoring is routinely practiced for CNI, it is not available for monitoring of anti-proliferative agents (MPA). Incidence of rejections may be considered an indicator of the adequacy of immunosuppression. However, the overall burden of immunosuppression is difficult to predict from biopsy registry alone and for which the incidence of infections also need to be taken into account. Complement-dependent cytotoxicity crossmatch and serological methods of HLA typing are routinely done at our institute. Newer modalities for cross-matching, single-antigen bead (SAB) assays and HLA typing (molecular methods) are not available in our setting as yet. KDIGO suggests the use of depleting agents like rATG in high-risk transplant. However, rATG is associated with increased risk of infections which are particularly pertinent to our population given the lower socioeconomic conditions in the majority of our patients. Some of the likely risk factors for acute rejection in our population include younger recipient age, prior blood transfusions, dialysis vintage, noncompliance to drugs due to financial constraints. All patients had undergone live, first renal transplantation with ABO-compatible donor and were mostly male recipients. These could be the reasons for equal or lower incidence of rejection within the 1styear of KT compared to that reported in some studies. The rates of rejections vary widely across studies partly because they depend on the expertise of the center in evaluating allograft biopsies.,,
We analyzed the biopsy findings in relation to various clinical presentations. Acute asymptomatic rise of SCr was the most common presentation. Majority of these were due to acute rejection, ATN, and acute CNI toxicity. Since acute CNI toxicity may not always have classical features, it is likely that some of the ATN could have been due to CNI toxicity. Most of the patients with ATN or acute CNI toxicity had the trough levels of CNI in acceptable range as per our protocol (not shown here). A small number had chronic changes in this group. This could have been due to an asymptomatic rise in SCr especially in the late post-KT period which was detected late and was considered acute in comparison to previous available SCr. CRAD was the second most common presentation. It is now known that CRAD can be immunological, non-immunological or both., In this study we found that chronic rejection and chronic CNI toxicity accounted for only about one fourth of CRAD. Some CRAD had only acute changes in the biopsy though the possibility of focal chronic changes missed on biopsy remains as such. These are likely to respond better to treatment than others. One of the cases had a c4d negative TG. A significant proportion of CRAD was due to GN with FSGS as the most common finding. However, the occurrence was GN late, i.e., >1 year post-KT. This is similar to the findings reported by RADR but much higher than that reported from other part of India. GN was also a major cause in those who presented with proteinuria and persistent active sediments. C4d negative TG was not reported as a part of chronic rejection in some cases. These were older reports when recognition of c4d negative AMR was not a included in Banff classification. Recurrent and de novo GN could not be differentiated as the NKD was not known in most of the patients. IGD was found in 9.09%, of which DGF occurred in 1.98% of cases and rejection in 2.47% within one week. The incidence of DGF is close to that found in other live donor programs.
The procedure of renal allograft biopsy was associated with minor complications in 3.17%. No major complication occurred. In comparison with that reported by other studies this was a low incidence.,
| Conclusion|| |
Renal allograft biopsy is the gold standard for diagnosis of RAD and is a safe procedure. The asymptomatic acute rise of SCr is a common presentation of RAD hence the importance of regular follow-ups and allograft biopsies (when indicated) cannot be over emphasized. CRAD in our setting can have myriad causes hence biopsy must be considered in these cases. Recurrent or de novo GN is an important cause of RAD in our population which may be due to the younger age of KTRs. Though newer modalities like SAB assay and molecular methods for HLA typing are not presently available in our institute, the current immunosuppression protocols have resulted in a comparable incidence of rejection. The limitations of this study include a relatively small number of patients, retrospective design of the study and role of protocol biopsy was not assessed. This being an observational study based on retrospective analysis a direct comparison with studies/data on other immuno-suppression and protocol biopsy protocols may not be applicable Despite these limitations the present study is an analysis and evaluation of the widely used transplantation protocol in wake of the updated Banff classification. Another strength of the study is that all biopsies were evaluated by single nephro-pathologist. It will also be helpful for analyzing time trends in live donor KT in future.
| Acknowledgment|| |
We thank all the residents of the Department of Nephrology, IPGMER and SSKM, Kolkata who have played a role in performing the biopsies as well as taking care of the patients.
Conflict of interest: None declared.
| References|| |
Morris P, Knechtle SJ. Kidney transplantation-principles and practice. Elsevier Health Sciences;2013Oct 24.
Jennette JC, Olson JL, Silva FG, D'Agati VD, eds. 7th
editionHeptinstall's Pathology of the Kidney: Lippincott Williams & Wilkins; 2015.
El-Zoghby ZM, Stegall MD, Lager DJ, Kremers WK, Amer H, Gloor JM, et al. Identifying specific causes of kidney allograft loss. Am J Transplant 2009;9:527-35.
Ranjan P, Nada R, Jha V, Sakhuja V, Joshi K. The role of C4d immunostaining in the evaluation of the causes of renal allograft dysfunction. Nephrol Dial Transplant 2008;23:1735-41.
Lo DJ, Kaplan B, Kirk AD. Biomarkers for kidney transplant rejection. Nat Rev Nephrol 2014;10:215-25.
Gondos A, Döhler B, Brenner H, Opelz G. Kidney graft survival in Europe and the United States: Strikingly different long-term outcomes. Transplantation 2013;95:267-74.
Rajapurkar MM, John GT, Kirpalani AL, Abraham G, Agarwal SK, Almeida AF, et al. What do we know about chronic kidney disease in India: First report of the Indian CKD registry. BMC Nephrol 2012;13:10.
Kazi JI, Mubarak M. Biopsy findings in renal allograft dysfunction in a live related renal transplant program. J Transplant Technol Res 2012;2(1):108.
Aryal G, Shah DS. Histopathological evaluation of renal allograft biopsies in Nepal: Interpretation and significance. J Pathol Nepal 2012;2:172-9.
Kostakis ID, Moris DN, Barlas A, Bokos I, Darema M, Theodoropoulou E, et al. Impact of donor and recipient age difference on long-term allograft survival after living donor renal transplantation: Analysis of 478 cases. Clin Transplant 2013;27:838-43.
Moosa MR. Impact of age, gender and race on patient and graft survival following renal transplantation – Developing country experience. S Afr Med J 2003;93:689-95.
Joosten SA, Sijpkens YW, van Kooten C, Paul LC. Chronic renal allograft rejection: Pathophysiologic considerations. Kidney Int 2005;68:1-3.
Chugh Kirpal S, Jha V. Problems and outcomes of living unrelated donor transplants in the developing countries. Kidney Int 2000;57: S131-5.
Uppin MS, Prayaga AK, Murty KV. Utility of renal allograft biopsy: An audit of 80 allograft biopsies. Indian J Nephrol 2013;23:155-6.
] [Full text]
Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immuno-staining methods in renal allograft biopsies. Clin J Am Soc Nephrol 2006;1:583-91.
Gourishankar S, Leduc R, Connett J, et al. Pathological and clinical characterization of the 'troubled transplant' : Data from the DeKAF study. Am J Transplant 2010;10:324-30.
Chapman JR, O'Connell PJ, Nankivell BJ. Chronic renal allograft dysfunction. J Am Soc Nephrol 2005;16:3015-26.
Kasiske BL, Kalil RS, Lee HS, Rao KV. Histopathologic findings associated with a chronic, progressive decline in renal allograft function. Kidney Int 1991;40:514-24.
Hariharan S, Adams MB, Brennan DC, et al. Recurrent and de novo glomerular disease after renal transplantation: A report from renal allo-graft disease registry (RADR). Transplantation 1999;68:635-41.
Philip KJ, Calton N, Pawar B. Nonrejection pathology of renal allograft biopsies: 10 years experience from a tertiary care center in North India. Indian J Pathol Microbiol 2011;54:700-5.
] [Full text]
Mengel M, Chapman JR, Cosio FG, et al. Protocol biopsies in renal transplantation: Insights into patient management and patho-genesis. Am J Transplant 2007;7:512-7.
Ahmad I. Biopsy of the transplanted kidney. Semin Intervent Radiol 2004;21:275-81.
Tsai SF, Chen CH, Shu KH, et al. Current safety of renal allograft biopsy with indication in adult recipients: An observational study. Medicine (Baltimore) 2016;95:e2816.
Dr. Smita Subhash Divyaveer
Department of Nephrology, Institute of Postgraduate Medical Education and Research and Seth Sukhlal Karnani Memorial Hospital, Kolkata
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]
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